Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B (NANB) viral hepatitis, infecting approximately 4 million people in the U.S. HCV is an enveloped RNA virus containing a single positive-stranded RNA genome. The viral protein NS5B is an RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication and represents a unique target for antiviral intervention. Although NS5B has been studied in vitro and its crystal structure determined, its molecular mechanism in HCV RNA replication in vivo is poorly understood. The recent development of a reverse genetics system for HCV provides the opportunity to perform genetic analysis of NS5B and other nonstructural viral proteins in HCV RNA replication. The overall goal of this research proposal is to determine the molecular basis underlying HCV RNA replication using biochemical and reverse genetic approaches. We have recently demonstrated that NS5B is able to initiate de novo RNA synthesis with purine but not pyrimidine nucleotides. In this study, we will determine the ribonucleoside triphosphate (NTP) binding site(s) of NS5B using chemical cross-linking and peptide sequence analysis. The biological roles of the NTP binding site(s) of NS5B will then be defined through mutagenesis analysis in conjunction with cross-linking analysis and in vitro RdRp assays for initiation and elongation of RNA synthesis. We have developed efficient cell culture systems for HCV replicon replication. The replicon replication systems will be used to determine the effects of mutations in the NTP binding sites on HCV RNA replication in the cell. As purified recombinant NS5B lacks template specificity in vitro, other nonstructural viral and cellular proteins may be required for specificity of HCV RNA replication in vivo. We will dissect the roles of NS5B and other nonstructural viral proteins in the control of HCV RNA replication by mutagenesis analysis coupled with reverse genetics approaches. These initial studies will lead to our long-term goal to understand the roles of the conserved genomic RNA sequences/structures and viral proteins in HCV RNA replication, virion assembly, and molecular pathogenesis. Information gained from these studies will provide not only meaningful insight to the molecular mechanisms of HCV RNA replication, but also novel antiviral targets for development of specific replicase inhibitors to treat HCV infection.